Protein glycosylation in eukaryotic cells is important for numerous cellular processes, including protein folding, lysosomal targeting, receptor signaling and cell-cell adhesion. Glycoproteins, therefore, are involved in most physiological processes, and aberrant protein glycosylation is observed in almost all major human diseases. Despite the pathophysiological significance of glycoproteins, the vast majority of them have not been characterized at the molecular level, and the functions of their glycans are poorly understood. Cell surface glycosylation is dynamic and changes in glycan structure accompany cell transformation in cancer progression. However, because of current limitations in molecular tools it is not yet possible to follow the dynamic changes of cell-surface glycans with high spatia and temporal resolution. The long-term objective of this project is to develop new imaging and proteomic tools to analyze changes in naturally- occurring glycans in disease-related processes. Our central hypothesis is that bioorthogonal click reactions with fast kinetics, high specificity ad biocompatibility can serve as the foundation upon which tools can be developed and applied to decipher the functional roles of glycans in human disease. In Aim 1, we will develop labeling methods based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) to incorporate small-molecule fluorescent probes on to cell-surface glycoconjugates. These methods will be combined with modified, stroboscopic, time-lapse imaging to achieve a fast form of localization microscopy for sub-diffraction-limit imaging of glycoconjugates in living systems. Methods will be developed to enable single molecule tracking of glycans on a specific membrane protein. Studies have shown that abnormal glycosylation in tumor cells is associated with cancer progression and malignancy by regulating adhesion, receptor signaling and protein expression. Terminal sialylation and fucosylation are key contributors to these processes. In Aim 2, we will develop a general glycoproteomic approach to identify sialylated glycoproteins in cancer cells. We will apply this approach for comparative analysis of the sialylated proteomes of cancer cells with distinct metastatic potentials. We hypothesize that abnormal glycosylation in tumor cells will affect the dynamic behaviors of membrane proteins, and thus adhesion and cancer metastasis. Glycoproteins identified from Aim 2 with unique expression patterns will be selected for further biological studies. We will alter glycosylation status of these proteins directly on th cell surface by in situ glycosylation reactions and by glycosidase treatment. The impact of the altered dynamics of the selected proteins on the membrane will be quantified, and the resulting influence, if any, upon cell adhesion and migration will be evaluated (Aim 3).